Interest in RNA-binding proteins (RNA-BPs) has burgeoned in recent years with the growing awareness that these proteins play a key role in a wide variety of cellular processes. However, very little is known about the structural features of these proteins that enable them to recognize their RNA targets with such high specificity. These investigations will focus on identifying and characterizing some of the features of RNA-BPs that are important for their ability to bind to specific RNAs. Particular attention will be devoted to protein paradigms representing three different families of RNA-BPs: the mammalian spliceosomal proteins U1A and U2B" (RRM family), the HIV-1 protein Rev (arginine-rich family), and the yeast regulatory protein Mer1 (KH family). The experimental strategy will emphasize genetic selection/screening of random mutations to identify important RNA-BP residues, followed by site-directed mutagenesis and biochemical analysis to better understand the role of these residues in RNA binding. The results of these studies will provide important insights into how members of three different RNA-BP families recognize their RNA targets, thereby shedding light on a key aspect of molecular biology that presently is poorly understood. This information will contribute to the goal of understanding how RNA-BPs in general are able to bind their targets with high affinity and specificity. In view of the central role that RNA-binding proteins play in the post-transcriptional control of gene expression, in HIV-1 infection, and in fragile X mental retardation, this knowledge should be of value for understanding mechanisms of pathogenesis and could eventually form the basis for novel gene therapies.